Structural Analysis of Biomolecules through a Combination of Mobility Capillary Electrophoresis and Mass Spectrometry

Abstract

The 3D structures of biomolecules determine their biological function. Established methods in biomolecule structure determination typically require purification, crystallization, or modification of target molecules, which limits their applications for analyzing trace amounts of biomolecules in complex matrices. Here, we developed instruments and methods of mobility capillary electrophoresis (MCE) and its coupling with MS for the 3D structural analysis of biomolecules in the liquid phase. Biomolecules in complex matrices could be separated by MCE and sequentially detected by MS. The effective radius and the aspect ratio of each separated biomolecule were simultaneously determined through the separation by MCE, which were then used as restraints in determining biomolecule conformations through modeling. Feasibility of this method was verified by analyzing a mixture of somatostatin and bradykinin, two peptides with known liquid-phase structures. Proteins could also be structurally analyzed using this method, which was demonstrated for lysozyme. The combination of MCE and MS for complex sample analysis was also demonstrated. MCE and MCE–MS would allow us to analyze trace amounts of biomolecules in complex matrices, which has the potential to be an alternative and powerful biomolecule structure analysis technique.

Structure and effective charge characterization of proteins by a mobility capillary electrophoresis based method

“…As depicted in Fig. 1, the hydrodynamic radius obtained from MCE experiments could be further utilized as a selection rule for MD simulations, so that the most probable protein conformation under experimental solvent conditions could be obtained 32,33. Briefly, a lot of possible conformations of a protein could be acquired from the MD simulation, and a selection rule is needed to pick out the most probable protein conformation.…”

“…As demonstrated in previous publications,32,33 MCE based experimental results could be used as a selection rule for MD simulations to predict the most probable protein structures in solution. However, the previous method requires the estimation of ion charge states before the ion hydrodynamic radius could be obtained.…”

“…In an MCE experiment as shown in Fig. 1, ions in a constant liquid flow will experience an electric field force 32. The constant liquid flow is maintained by applying a constant pressure over the separation channel.…”

“…Recently, mobility capillary electrophoresis (MCE) was proposed to achieve ion separation and hydrodynamic radius measurement simultaneously 32,33. In MCE experiments, ions were driven in a constant flow34,35 and separated by an electric field, making MCE a liquid-phase analogue of IMS.…”

“…However, salts are present in many practical biosystems, and their presence may help in maintaining the native conformations of proteins. Furthermore, the accuracy of protein charge state estimation also limits the accuracy of ion hydrodynamic radius calculation 32,33. In fact, the protein charge state is an important factor affecting its activity and solubility,39 and it may affect the protein conformation in turn 40–42.…”